Journal: Nature

Article Title: Low-dose metformin targets the lysosomal AMPK pathway through PEN2

doi: 10.1038/s41586-022-04431-8

Figure Lengend Snippet: a , Serum metformin concentrations in human subjects. Serum samples were collected at indicated time points from subjects after taking 0.5 g of Metformin Hydrochloride Extended-release Tablets. Data are shown as mean ± s.e.m.; n = 6. b , c , e , Low metformin activates AMPK in primary hepatocytes without elevating AMP. Human ( b ) or mouse ( c , e ) primary hepatocytes were treated with metformin (Met), or PBS (Saline) for 2 h, and the levels of p-AMPKα and p-ACC ( b , c ), as well as the AMP:ATP and ADP:ATP ratios ( b , e ) were determined [shown as mean ± s.e.m.; n = 5 ( b ) or 4 ( e ) cells for each condition, and P value by two-sided Student’s t -test ( b ), or one-way ANOVA followed by Sidak ( e )]. d , Metformin inhibits v-ATPase in purified lysosomes. Lysosomes purified from mouse livers were incubated with 5 μM metformin for 1 h. The activity of v-ATPase was determined by the rates to hydrolyse ATP (left panel) and to transport protons (right panel). Data are shown as mean ± s.e.m.; n = 3; P value by two-sided Student’s t -test. f , s , Low metformin does not affect mitochondrial membrane potential. Mouse primary hepatocytes ( f ), MEFs ( s , left panel), or HEK293T cells ( s , right panel) were treated with 5 μM metformin for 2 h ( f ), 200 μM metformin for 12 h ( s , left panel) or 300 μM metformin for 12 h ( s , right panel) (higher concentrations and longer treatment times were used in MEFs and HEK293T cells because of lack of OCTs), and were loaded with JC-1 dye for another 30 min. After normalisation to the group without metformin treatment, the data are shown as mean ± s.e.m.; n = 37 (control) and 34 (metformin-treated) with mouse primary hepatocytes, n = 36 (control) and 40 (metformin-treated) for MEFs, and n = 35 (control) and 36 (metformin-treated) with HEK293T cells; and P value by two-sided Mann-Whitney ( f and s , left panel) or two-sided Student’s t -test ( s , right panel). g , t , Low metformin does not affect mitochondrial respiration. Mouse primary hepatocytes ( g , approximately 3,000 cells in total), MEFs ( t , left panel, approximately 10,000 cells in total), or HEK293T cells ( t , right panel, approximately 10,000 cells in total) were treated as in f , left panel of s , and right panel of s . ATP production-coupled OCR was determined by subtracting basal OCR from that treated with 10 μM oligomycin. Data are mean ± s.e.m.; n = 6 with hepatocytes and MEFs, and n = 4 with HEK293T cells; and P value by two-sided Student’s t -test. h , i , l , Mice taking 1 g/l metformin from drinking water resembles the situation of human patients taking standard clinical doses of metformin. As depicted in i , mice at 4-week old were treated with metformin in drinking water for 7 days. At day 8, mice were sacrificed at indicated times of the day. The mice were then divided into two groups, one for sacrifice to collect serum, and the others for the liver tissue. Results are mean ± s.e.m.; n = 5 for each time point, except n = 4 for the 2 g/l group at 0:00, 4:00 and 18:00. Note that perhaps owing to the bitterness of metformin at higher doses (10 g/l), some of the mice showed a decreased water intake (hence metformin), and larger variations of the serum metformin concentrations than those of 1 g/l and 2 g/l were observed. j , k , m , n , High doses of metformin leads to increased AMP/ADP levels, and bypasses the requirement of PEN2 for AMPK activation. Mice were treated as in i , followed by analysis of p-AMPKα and p-ACC ( j , m , n ) and hepatic AMP:ATP and ADP:ATP ratios, the absolute concentrations of AMP, ADP and ATP, and the hepatic metformin concentrations. Results are mean ± s.e.m.; n = 5 ( k , m ) and n = 16 ( n ) for each treatment, and P value by one-way ANOVA followed by Dunn ( k ) or Tukey ( m ). Isc; hepatic ischemia (for 5 sec). Note that in m , n , readouts were determined in the liver from the mice that did not undergo the step of blood draining (different from h ), because ischemia will increase AMP and ADP, and will cause AMPK activation unrelated to the lysosomal pathway . The legitimacy for skipping the step of blood draining was based on the observation that hepatic metformin concentration is similar to that in the serum in our animal setting, as shown in h - the residual blood would not significantly interfere with the readout of the hepatic metformin concentration. o , p , v , w , AMPK can be activated in MEFs and HEK293T cells in AMP/ADP-independent manner in low metformin. MEFs ( o ), HEK293T cells ( p ), and the OCT1-expressing MEFs ( v ) and HEK293T cells ( w ) were treated with metformin at indicated concentrations for 12 h ( o , p ) or 2 h ( v , w ), followed by analysis of intracellular metformin concentrations [shown as mean ± s.e.m.; n = 4 (for each metformin concentration in o , p and w , except n = 3 for the 0.2 mM metformin in o and p ) or 5 ( v )], p-AMPKα and p-ACC, and AMP:ATP, and ADP:ATP ratios [shown as mean ± s.e.m.; n = 4 (for each metformin concentration in o and p , except n = 3 for the ratios at 5 mM metformin in o ) or 5 ( v , w ); and P values by one-way ANOVA, followed by Sidak ( o , and AMP:ATP of p ), Tukey (ADP:ATP of p , and w ), or Dunn ( v )], as well as the absolute concentrations of AMP, ADP and ATP. q , r , Low metformin deacidifies lysosomes. MEFs ( q ) and HEK293T cells ( r ) pre-labelled with LysoSensor Green DND-189 and Hoechst were treated as in s . Representative images are shown (left panel); the relative fluorescent intensities of Lysosensor (normalised to the intensity of Hoechst) are shown on the right. Results are mean ± s.e.m.; n = 28 (control) and 27 (metformin-treated) from 3 dishes/experiments for MEFs, and n = 21 (control) and 20 (metformin-treated) from 3 dishes/experiments for HEK293T cells; and P value by two-sided Mann-Whitney test. u , Metformin is not accumulated in mitochondria. MEFs were treated as in s , and metformin concentrations in mitochondria and cytosol fractions (normalised to protein concentration) are shown as mean ± s.e.m.; n = 4, and P value by two-sided Student’s t -test. Experiments in this figure were performed three times, except c , h , j , k and o four times.

Article Snippet: During imaging, live cells were kept at 37 °C, 5% CO 2 in a humidified incubation chamber (Zeiss, Incubator PM S1).

Techniques: Saline, Purification, Incubation, Activity Assay, Membrane, Control, MANN-WHITNEY, Activation Assay, Concentration Assay, Expressing, Protein Concentration

Journal: Nature

Article Title: Low-dose metformin targets the lysosomal AMPK pathway through PEN2

doi: 10.1038/s41586-022-04431-8

Figure Lengend Snippet: a , Synthesis and purification of photoactive metformin probes (Met-Ps). Reactions for conjugating 3-(but-3-yn-1-yl)-3-(2-iodoethyl)-3H-diazirine to metformin that introduced a diazirine with a terminal alkyne moiety at either the N4 or N1/N2 position of metformin yields two types of Met-P (Met-P1 and Met-P2) products (upper panel). The two products were further separated on a preparative HPLC (lower panel). See detailed procedures, HSMS data, and NMR data in Methods section and Supplementary Fig. . b , Met-P1 is able to inhibit v-ATPase. Lysosomes purified from MEFs were incubated with the two Met-Ps at 10 μM for 1 h. The activity of v-ATPase was determined by its rate to hydrolyse ATP as in Extended Data Fig. . After normalisation to the group without Met-P added, the data are shown as mean ± s.e.m.; n = 3 for each condition, and P value by one-way ANOVA, followed by Dunnett. c , Reactions taking place to form the Met-P1 and proteins conjugates. First, proteins were incubated with Met-P1. The metformin probe-protein mixture was exposed to UV light, followed by addition of Cu(II) salt, which catalyses a [3 + 2] azide-alkyne cycloaddition with biotin-azide, thus biotinylating probe-target complexes, allowing for the pull down of such complexes with NeutrAvidin beads. d , Interaction between PEN2 and metformin probe. HEK293T cells transfected with HA-tagged PEN2 were lysed. Total cell lysates (TCL) were incubated with 10 μM Met-P1, and subsequent exposure to UV, and were then mixed with 1 mM biotin-N 3 linker. The biotinylated proteins were then affinity-pulldown (AP) by NeutrAvidin beads, followed by immunoblotting with antibody against HA tag. Experiments in this figure were performed three times, except a seven times.

Article Snippet: During imaging, live cells were kept at 37 °C, 5% CO 2 in a humidified incubation chamber (Zeiss, Incubator PM S1).

Techniques: Purification, Incubation, Activity Assay, Transfection, Western Blot

Journal: Nature

Article Title: Low-dose metformin targets the lysosomal AMPK pathway through PEN2

doi: 10.1038/s41586-022-04431-8

Figure Lengend Snippet: a , A schematic depicting the procedure of the affinity-based approach that used a photoactive metformin probe (Met-P) to identify target(s) of metformin from protein extracts of lysosomes purified from MEFs. MS, mass spectrometry. b , c , Knockout of Pen2 blocks the activation of AMPK by low-dose metformin. Mouse primary hepatocytes ( b ) and MEFs ( c ; clone 1, and same hereafter, unless stated otherwise) were treated with 5 μM and 200 μM metformin for 2 h and 12 h, respectively, followed by analysis of p-AMPKα and p-ACC. WT, wild type. d , e , STORM image of MEFs ( d ) and TEM image of HEK293T cells ( e ) showing that a portion of PEN2 is localized to the lysosome ( e , black arrowheads) and overlaps with the lysosome marker LAMP2 ( d ). f , g , PEN2 is able to bind metformin. f , In SPR assays, PEN2 was incubated with metformin at the indicated concentrations. g , In Met-P1-binding assays, HEK293T cells transfected with PEN2 or PEN2-2A were lysed, incubated with 10 μM Met-P1 and then biotinylated, and then affinity pull-down (AP) of biotinylated proteins was performed. TCL, total cell lysate. h , PEN2-2A does not mediate AMPK activation by metformin. Pen2 –/– MEFs re-introduced with haemagglutinin (HA)-tagged PEN2-2A were treated with 200 μM metformin for 12 h, followed by analysis of p-AMPKα and p-ACC. For gel source data, see Supplementary Fig. . Experiments in this figure were performed three times, except those in b and c , which were performed four times.

Article Snippet: During imaging, live cells were kept at 37 °C, 5% CO 2 in a humidified incubation chamber (Zeiss, Incubator PM S1).

Techniques: Purification, Mass Spectrometry, Knock-Out, Activation Assay, Marker, Incubation, Binding Assay, Transfection

Journal: Nature

Article Title: Low-dose metformin targets the lysosomal AMPK pathway through PEN2

doi: 10.1038/s41586-022-04431-8

Figure Lengend Snippet: a , b , Knockdown of PEN2 impairs the activation of AMPK, and inhibition of v-ATPase by metformin. MEFs infected with lentivirus carrying two distinct siRNAs (#1 or 2#) against PEN2 , or GFP as a control, were treated with 200 μM metformin for 12 h, representative images of the experiments shown in a upper, followed by analysis of the lysosomal pH [ a lower, shown as mean ± s.e.m., n = 20 (control) and 21 (metformin-treated) cells for si GFP , n = 25 cells for si PEN2 #1, and n = 29 (control) and 22 (metformin-treated) cells for si PEN2 #2, all from 2 dishes/experiments; and P value by two-way ANOVA, followed by Tukey] and the determination of p-AMPKα and p-ACC ( b ). c , Knockout of PEN2 abrogates the inhibition of v-ATPase by metformin in mouse primary hepatocytes (left panel), MEFs (middle panel), and HEK293T cells (right panel). MEFs, HEK293T cells were treated with 200, 300 μM metformin for 12 h, mouse primary hepatocytes were treated with 5 μM metformin for 2 h, and then labelled with Lysosensor, along with Hoechst. The lysosomal pH was determined as in Fig. . Data are shown as mean ± s.e.m., n = 26 (control) and 31 (metformin-treated) from 6 dishes/experiments for primary hepatocytes, n = 25 (control) and 22 (metformin-treated) from 4 dishes/experiments for MEFs, and n = 30 (control) and 29 (metformin-treated) from 6 dishes/experiments for HEK293T cells; P value within each cell type was determined by two-sided Student’s t -test. d , g , h , k , Knockout of PEN2 blocks AMPK activation by low metformin. Clone #2 of PEN2 -/- MEFs ( d ) treated with 200 μM (low concentration) metformin, or clone #1 and clone #2 of PEN2 -/- HEK293T cells ( g and h ), treated with 300 μM (low concentration for the cell line) metformin, or OCT1-expressing PEN2 -/- MEFs and HEK293T cells treated with 5 μM (low concentration) metformin ( k ) or 5 mM (high concentration, as a control for d , g and h ), 500 μM metformin (high concentration, for k ), for 12 h ( d , g and h ) or 2 h ( k ), were subjected to immunoblotting for the analysis of p-AMPKα and p-ACC. See also results with clone #1 of PEN2 -/- MEFs in Fig. . e , f , Strategies to generate MEFs ( e ) and HEK293T cells ( f ) with knockout of PEN2 . Two distinct sets of sgRNAs for each cell line, whose sequences are listed in Methods section, were applied to generate PEN2 -/- cells. Two clones (#1 and #2) for each cell line type were established. i , Knockout of PEN2 blocks the inhibition of v-ATPase by metformin in purified lysosomes. Lysosomes purified from PEN2 -/- MEFs were incubated with 5 μM metformin for 1 h. The activity of v-ATPase was determined as in Extended Data Fig. . Data are shown as mean ± s.e.m.; n = 3 for each condition, and P value by two-sided Student’s t -test. j , Knockout of PEN2 does not affect metformin uptake. Mouse primary hepatocytes, MEFs and HEK293T cells were treated as in c , followed by determining intracellular metformin concentrations. Data are shown as mean ± s.e.m., n = 4 for each genotype, and P value within each cell type by two-sided Student’s t -test. l , Re-introduction of PEN2 into PEN2 -/- MEFs or HEK293T cells restores AMPK activation. PEN2 -/- MEFs (left panel) or HEK293T cells (right panel) were infected with lentiviruses expressing HA-tagged PEN2 (all expressed at close-to-endogenous levels driven by pBOBI vector). Cells were treated with 200 or 300 μM (low concentration), or 5 mM (high concentration, as a control) metformin for 12 h, followed by analysis of p-AMPKα and p-ACC. m , Activity of the γ-secretase holoenzyme is dispensable for metformin-induced AMPK activation. MEFs were treated with DAPT (left panel) or RO4929097 (RO, right panel) at indicated concentrations for 12 h or 48 h. Twelve hours before lysis, cells were treated with 200 μM metformin, then lysed for analysis of p-AMPKα and p-ACC. n , Loss of APH1 does not affect metformin-induced activation of AMPK. MEFs with APH1A , APH1B and APH1C triple knockout were treated with 200 μM or 5 mM metformin for 12 h, followed by analysis of p-AMPKα and p-ACC. o , Strategies to generate MEFs with knockout of nicastrin. sgRNAs against NCSTN , whose sequences are listed in Methods section, were applied to generate NCSTN -/- MEFs. p , q , Knockout of NCSTN , through decreasing the protein levels of PEN2, impairs metformin-induced activation of AMPK. NCSTN -/- MEFs ( p ) or NCSTN -/- MEFs with HA-tagged PEN2 expressed ( q , expressed at close-to-endogenous levels driven by the lentiviral system using pBOBI vector, as validated in r ) were treated with 200 μM (low concentration) or 5 mM (high concentration, as a control) metformin for 12 h, followed by analysis of p-AMPKα and p-ACC. r , Protein levels of PEN2 in MEFs with knockout of NCSTN . Cells were lysed for analysis of PEN2 protein levels by immunoblotting, followed by densitometry analysis. s , Strategies to generate MEFs with knockout of presenilins. sgRNAs against PS1 (left panel) and PS2 (right panel), whose sequences are listed in Methods section, were applied to generate PS1 - or PS2 -KO MEFs. Experiments in this figure were performed three times, except b , h , l , four times.

Article Snippet: During imaging, live cells were kept at 37 °C, 5% CO 2 in a humidified incubation chamber (Zeiss, Incubator PM S1).

Techniques: Knockdown, Activation Assay, Inhibition, Infection, Control, Knock-Out, Concentration Assay, Expressing, Western Blot, Clone Assay, Purification, Incubation, Activity Assay, Plasmid Preparation, Lysis, Triple Knockout

Journal: Nature

Article Title: Low-dose metformin targets the lysosomal AMPK pathway through PEN2

doi: 10.1038/s41586-022-04431-8

Figure Lengend Snippet: a , Incubation with metformin decreases the thermal transition midpoint (Tm) of PEN2. FLAG-tagged PEN2 was ectopically expressed in HEK293T cells and purified. Some 10 μM of the purified PEN2 was then incubated with 10 μM metformin in PBS buffer, followed by determining the Tm on a differential scanning calorimetre. Enthalpy changes of PEN2, and PEN2 incubated with metformin at indicated temperatures are shown. b , ITC assay showing that PEN2 is able to bind metformin. Metformin (2 mM stock concentration) was loaded stepwise to 60 μM PEN2 (purified as in a ) in PBS buffer. Integrated data (lower panel) were obtained by fitting raw data (upper panel) with the two sets of sites model. c , Unlike PEN2, other γ-secretase subunits do not bind metformin. HEK293T cells transfected with HA-tagged PS1 (either its NTD or CTD), PS2 (either its NTD or CTD), NCSTN, APH1A, APH1B, APH1C, or PEN2 as a control, were lysed, followed by incubation with 10 μM Met-P1, exposure to UV, and were then mixed with 1 mM biotin-N 3 linker. The biotinylated proteins were then pulled down by NeutrAvidin beads, followed by immunoblotting with antibody against HA tag. d , Determination of the binding sites of PEN2 for metformin by mass spectrometry. HEK-293T cells expressing HA-tagged PEN2 were lysed, followed by incubation with 10 μM Met-P1, exposure to UV, and the potential modified residues (conjugated with biotinylated Met-P1, with an increase of m/z by 222.17) were determined by mass spectrometry, revealing two Met-P1-conjugated residues, Y47 and Y91, as shown by the typical spectrograms, with Y91 conjugated at a much lower efficiency. e , In silico modelling of metformin bound to the N-terminal, cytosolic face of PEN2. Modelling was performed according to the reported cryo-electron microscopy structure (PDB ID: 6IYC). As shown in this figure, metformin could be protonated by carboxyl groups from residue E40 of PEN2, and then be docked onto PEN2 via salt bridges formed between N2-E40 (position of metformin to the residue of PEN2, and the same below) (2.3 Å) and N4-E40 (2.3 Å). Furthermore, two potential hydrogen bonds formed between N1-F35 (2.0 Å), N2-W36 (2.7 Å) may further strengthen the interaction between metformin and PEN2. f , PEN2-2A fails to bind metformin. PEN2-2A (F35A and E40A, purified as in a ) was immobilised on a BIAcore CM5 sensor chip, followed by analysing its interaction with metformin by an SPR assay as in Fig. . Sensorgrams of each measurement are shown. See also sensorgrams from another two repeats of Fig. below. g , Validation data showing that re-introduced PEN2 and its mutants are expressed at a close-to-endogenous level in PEN2 -/- MEFs and HEK293T cells. h , PEN2-2A fails to mediate the effect of metformin on v-ATPase inhibition. MEFs were treated as in Extended Data Fig. , followed by analysis of lysosomal pH as in Fig. . After normalisation to the group without metformin treatment within each genotype (same hereafter), results are shown as mean ± s.e.m.; n = 20 (control) and 23 (metformin-treated) from 4 dishes/experiments for PEN2-WT, and n = 20 (normal) and 23 (metformin-treated) from 6 dishes/experiments for PEN2-2A; and P value within each genotype was determined by two-sided Student’s t test with Welch’s correction (for re-introduction of wild type PEN2) or by two-sided Student’s t test (for re-introduction of PEN2-2A). i , PEN2-2A mutant retains proper subcellular localisation as wildtype PEN2. PEN2 -/- MEFs were infected with lentivirus expressing HA-tagged PEN2-2A or wildtype PEN2. Cells were then stained with mouse anti-HA antibody and rat anti-LAMP2 antibody, followed by incubation with Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 donkey anti-rat IgG secondary antibodies (upper panel), or mouse anti-HA antibody and Alexa Fluor 594 goat anti-mouse IgG secondary antibody, followed with Alexa Fluor 488-conjugated rabbit anti-PDI antibody (lower panel). Mander’s overlap coefficients are plotted as mean ± s.e.m., n = 20 for each genotype, with P values calculated by two-sided Student’s t -test. The areas defined by dashed boxes on each representative image are enlarged as insets. j , In silico modelling of metformin bound to the C-terminal of PEN2. Residues D90 with the top-ranked score, as well as salt bridges and hydrogen bonds formed, are shown. k , PEN2-D90A displays full affinity for metformin. HEK293T cells transfected with HA-tagged PEN2-D90A or wild type PEN2 were lysed, followed by incubation with 10 μM Met-P1, exposure to UV, and were then mixed with 1 mM biotin-N 3 linker. The biotinylated proteins were then pulled down by NeutrAvidin beads, followed by immunoblotting with antibody against HA tag. l , Residue D90 in PEN2 is not involved in metformin-induced AMPK activation. PEN2 -/- MEFs re-introduced with HA-tagged PEN2-D90A or wild type PEN2 (both were expressed at close-to-endogenous levels) were treated with 200 μM (low concentration) or 5 mM (high concentration, as a control) metformin for 12 h, followed by analysis of p-AMPKα and p-ACC. m , o , Effects of PEN2 mutant and metformin on the activity of γ-secretase. HEK293T cells ( o ) or PEN2 -/- HEK293T cells ( m ) were infected with lentivirus expressing Myc-tagged NotchΔE (NΔE). In m , cells were also infected with lentivirus expressing HA-tagged PEN2-2A, PEN2-20A or wildtype PEN2, in addition to the Myc-tagged NotchΔE (NΔE). Cells were then treated with 300 μM metformin for 12 h ( o ), or 100 μM DAPT for 12 h, the inhibitor to γ-secretase as a control ( m ). The cleavage of NotchΔE was determined by the protein levels of its NICD domain by immunoblotting. LE, long exposure; SE, short exposure. n , The PEN2 mutations or metformin do not affect the complex formation of γ-secretase. PEN2 -/- MEFs infected with lentivirus expressing HA-tagged PEN2-2A, PEN2-20A (residues 27, 28, 30, 31, 34, 38, 42, 43, 57, 58, 60, 63 to 65, 67, 68, 71, 72, 74 and 75 of PEN2 mutated to alanine, see Extended Data Fig. ) or wildtype PEN2, were treated with 200 μM metformin for 12 h and lysed, followed by immunoprecipitation (IP) with antibodies against HA. Immunoprecipitants were than subjected to immunoblotting with antibodies against PS1, PS2, NCSTN, APH1A/B/C, as well as HA (PEN2). Experiments in this figure were performed three times, except g , k and l four times.

Article Snippet: During imaging, live cells were kept at 37 °C, 5% CO 2 in a humidified incubation chamber (Zeiss, Incubator PM S1).

Techniques: Incubation, Purification, Isothermal Titration Calorimetry, Concentration Assay, Transfection, Control, Western Blot, Binding Assay, Mass Spectrometry, Expressing, Modification, In Silico, Cryo-Electron Microscopy, Residue, SPR Assay, Biomarker Discovery, Inhibition, Mutagenesis, Infection, Staining, Activation Assay, Activity Assay, Immunoprecipitation

Journal: Nature

Article Title: Low-dose metformin targets the lysosomal AMPK pathway through PEN2

doi: 10.1038/s41586-022-04431-8

Figure Lengend Snippet: a – c , Identification of ATP6AP1 as an interacting protein of PEN2. Lysates of HEK293T cells expressing HA–PEN2 or Myc–ATP6AP1 ( a ), and lysates from wild-type MEFs, Pen2 –/– MEFs ( b ) or Atp6ap1 –/– MEFs ( c ) were incubated with 10 μM metformin and immunoprecipitated (IP) for the PEN2 and AP1 proteins. d , Metformin does not promote the interaction between ATP6AP1 and PEN2-20A. HEK293T cells transfected with HA-tagged PEN2 or PEN2-20A were lysed and treated as in a . The interaction between ATP6AP1 and PEN2 was analysed by IP followed by IB. e–i , Loss of the PEN2–ATP6AP1 interaction abolishes the effects of metformin on AMPK activation. Atp6ap1 –/– MEFs re-introduced with ATP6AP1 Δ420–440 ( e ) or Pen2 –/– MEFs re-introduced with the PEN2-20A mutant ( f ) were treated with 200 μM metformin for 12 h followed by analysis of p-AMPK and p-ACC. g – i , The effects of ATP6AP1 and PEN2 mutants on the lysosomal translocation of AXIN ( g ), and the formation of the AXIN-based complex ( h , i ) were analysed. Concanamycin A (conA; 5 μM for 2 h) was used as a control. FL, full length. j , A schematic depicting that the metformin–PEN2–ATP6AP1 and the FBP–aldolase axes constitute two incoming shunts that converge at v-ATPase to elicit AMPK activation through the lysosomal pathway. For gel source data, see Supplementary Fig. . Data are the mean ± s.e.m., n values are labelled on each panel, and P values were calculated using two-sided Student’s t -test ( a , for Myc–ATP6AP1), two-sided Student’s t -test with Welch’s correction ( a , for HA–PEN2) or two-way ANOVA, followed by Tukey’s test ( g ). Experiments in this figure were performed three times, except for a (four times), and h and i (five times).

Article Snippet: During imaging, live cells were kept at 37 °C, 5% CO 2 in a humidified incubation chamber (Zeiss, Incubator PM S1).

Techniques: Expressing, Incubation, Immunoprecipitation, Transfection, Activation Assay, Mutagenesis, Translocation Assay, Control

Journal: Nature

Article Title: Low-dose metformin targets the lysosomal AMPK pathway through PEN2

doi: 10.1038/s41586-022-04431-8

Figure Lengend Snippet: a , PEN2 interacts with ATP6AP1. HEK293T cells were transfected with HA-tagged PEN2 and Myc-tagged ATP6AP1 (AP1). Cells were lysed, and 10 μM metformin (final concentration) or PBS was added to the lysates. Immunoprecipitation (IP) was performed using antibodies against HA, followed by immunoblotting with antibodies indicated. b , PEN2 interacts with ATP6AP1 in vitro . Some 1 μg of FLAG-tagged PEN2 (expressed in HEK293T cells, and purified through eluting with FLAG® peptide) were incubated with 1 μg of Strep-tagged ATP6AP1 (expressed in HEK293T cells, and purified through eluting with desthiobiotin) (input) in lysis buffer, then with metformin at indicated concentrations for 2 h. Immunoprecipitation was performed using ANTI-FLAG® M2 Affinity Gel, followed by immunoblotting with antibodies indicated. c , Domain mapping for the region on ATP6AP1 responsible for PEN2-binding. HA-tagged PEN2 was co-transfected with Myc-tagged ATP6AP1, or its deletion mutants into HEK293T cells. Immunoprecipitation was performed using antibody against Myc-tag, followed by immunoblotting with antibodies indicated. d , Replacement of ATP6AP1 transmembrane domain with that of LAMP2, blocks its interaction with PEN2. HEK293T cells transfected with HA-tagged PEN2-D90A, along with Myc-tagged LAMP2 TM -ATP6AP1 or wildtype ATP6AP1, were lysed, and 10 μM metformin was added to the lysates, followed by immunoprecipitation with antibody against HA, and immunoblotting with antibodies indicated. e , In silico modelling of ATP6AP1 (cyan) bound to PEN2 (magenta). Circled area indicates the predicted interface, in which residues 27, 28, 30, 31, 34, 38, 42, 43, 57, 58, 60, 63 to 65, 67, 68, 71, 72, 74 and 75, within the transmembrane domain of PEN2, are involved. f , ATP6AP1 shows much weaker interaction with other γ-secretase subunits than PEN2. MEFs were lysed and incubated with metformin as in a , followed by immunoprecipitation with antibodies against ATP6AP1, or PEN2 as a control. Immunoprecipitants were than subjected to immunoblotting with antibodies against PS1, PS2, NCSTN, APH1A/B/C, as well as PEN2 and ATP6AP1. g , Metformin, through promoting the association between PEN2 and ATP6AP1, enhances association of ATP6AP1 and γ-secretase. PEN2 -/- MEFs infected with lentivirus expressing HA-tagged PEN2 or its 20A mutant (lacking the interface for ATP6AP1) were lysed and incubated with metformin as in a , followed by immunoprecipitation with antibodies against ATP6AP1. Immunoprecipitants were than subjected to immunoblotting with antibodies against PS1, PS2, NCSTN, APH1A/B/C, as well as PEN2 and ATP6AP1. h , ATP6AP1 does not interact with metformin. HEK293T cells transfected with HA-tagged ATP6AP1, or HA-tagged PEN2 as a control, were lysed, followed by analysing the interaction between ATP6AP1 or PEN2 with Met-P1 as in Extended Data Fig. . i , Strategies to generate MEFs (lower panel) or HEK293T cells (upper panel) with knockout of ATP6AP1 . sgRNAs against ATP6AP1 , whose sequences are listed in Methods section, were applied to generate ATP6AP1 -/- MEFs and HEK293T cells. j , Knockout of ATP6AP1 leads to constitutive activation of AMPK. MEFs with ATP6AP1 knocked out, along with its wildtype control, were incubated with metformin at indicated concentrations for 12 h, followed by analysing p-AMPK and p-ACC. k , Knockout of ATP6AP1 renders v-ATPase inactive. ATP6AP1 -/- MEFs were treated with 200 μM metformin for 12 h, followed by analysis of lysosomal pH with the Lysosensor dye. Data (relative intensity of Lysosensor, processed as in Fig. ) were graphed as mean ± s.e.m., n = 29 (control) and 26 (metformin-treated) cells from 6 dishes/experiment for WT MEFs, and 28 (control) and 23 (metformin-treated) cells from 4 dishes/experiments for ATP6AP1 -/- MEFs, P value within each genotype was determined by two-sided Mann-Whitney test (for WT MEFs), or by two-sided Student’s t -test (for ATP6AP1 -/- MEFs). l , Validation data showing that the re-introduced ATP6AP1 and its mutants are expressed at a close-to-endogenous level in ATP6AP1 -/- MEFs (upper panel) and HEK293T cells (lower panel). Experiments in this figure were performed three times, except b , f , h , four times and l five times.

Article Snippet: During imaging, live cells were kept at 37 °C, 5% CO 2 in a humidified incubation chamber (Zeiss, Incubator PM S1).

Techniques: Transfection, Concentration Assay, Immunoprecipitation, Western Blot, In Vitro, Purification, Incubation, Lysis, Binding Assay, In Silico, Control, Infection, Expressing, Mutagenesis, Knock-Out, Activation Assay, MANN-WHITNEY, Biomarker Discovery

Journal: Nature

Article Title: Low-dose metformin targets the lysosomal AMPK pathway through PEN2

doi: 10.1038/s41586-022-04431-8

Figure Lengend Snippet: a , b , The truncated ATP6AP1 mutant (ATP6AP1 Δ420-440 ) or the chimeric construct LAMP2 TM -ATP6AP1, which are able to maintain the basal activity of v-ATPase, fails to mediate metformin to inhibit v-ATPase. ATP6AP1 -/- MEFs re-introduced with full length (FL) ATP6AP1, ATP6AP1 Δ420-440 or LAMP2 TM -ATP6AP1 (all expressed at close-to-endogenous levels) were treated with 200 μM metformin for 12 h (or 5 μM conA for 2 h as a control, right panel of a ), followed by analysis of lysosomal pH by labelling cells with Lysosensor, along with Hoechst. Data (relative intensity of Lysosensor, processed as in Fig. ) were graphed as mean ± s.e.m., n = 20 cells from 3 dishes/experiment ( a ) and n = 28 (control) and 23 (metformin-treated) for ATP6AP1 -/- MEFs re-introduced with full length ATP6AP1, and n = 26 (control) and n = 25 (metformin-treated) for ATP6AP1 -/- MEFs re-introduced with ATP6AP1 Δ420-440 , from 4 dishes/experiment ( b ) for each genotype/treatment. Results are mean ± s.e.m.; P value was determined by one-way ANOVA followed by Dunn ( a , left panel) or by two-sided Student’s t test with Welch’s correction ( a , right panel) or by two-sided Mann-Whitney test ( b ). c , ATP6AP1 Δ420-440 mutant fails to mediate the effects of metformin on AMPK activation. ATP6AP1 -/- MEFs (left panel) or HEK293T cells (right panel) stably expressing Myc-tagged OCT1 were re-introduced with full length (FL) ATP6AP1 or its Δ420-440 mutant (expressed at close-to-endogenous levels). Cells were treated with 5 μM metformin or 500 μM metformin (high concentration, as a control) for 2 h, followed by analysis of p-AMPK and p-ACC. d , ATP6AP1 mutant that cannot interact with PEN2 fails to mediate AMPK activation by metformin. ATP6AP1 -/- MEFs re-introduced with full length ATP6AP1 or LAMP2 TM -ATP6AP1 (expressed at close-to-endogenous levels) were treated with 200 μM metformin for 12 h, followed by analysis of p-AMPKα and p-ACC by immunoblotting. e , PEN2-20A mutant retains proper subcellular localisation. PEN2 -/- MEFs were infected with lentivirus expressing HA-tagged PEN2-20A or wildtype PEN2. Cells were then stained with mouse anti-HA antibody and rat anti-LAMP2 antibody, followed by incubation with Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 donkey anti-rat IgG secondary antibodies (upper panel), or mouse anti-HA antibody and Alexa Fluor 594 goat anti-mouse IgG secondary antibody, and subsequently with Alexa Fluor 488-conjugated rabbit anti-PDI antibody (lower panel). Mander’s overlap coefficients are plotted as mean ± s.e.m., n = 20 for each genotype, with P values calculated by two-sided Student’s t -test. The areas defined by dashed boxes on each representative image are enlarged as insets. f , PEN2 -/- MEFs re-introduced with wildtype PEN2 or PEN2-20A mutant were treated like Extended Data Fig. , followed by analysis of the lysosomal pH [shown as mean ± s.e.m., n = 29 (control) and 27 (metformin-treated) from 6 dishes/experiments for PEN2 -/- MEFs re-introduced with wildtype PEN2, n = 20 (control) and 23 (metformin-treated) from 4 dishes/experiments for PEN2 -/- MEFs re-introduced with PEN2-20A; and P value within each genotype by two-sided Student’s t test with Welch’s correction (wildtype PEN2) or two-sided Student’s t test (PEN2-20A)]. Experiments in this figure were performed three times, except d four times.

Article Snippet: During imaging, live cells were kept at 37 °C, 5% CO 2 in a humidified incubation chamber (Zeiss, Incubator PM S1).

Techniques: Mutagenesis, Construct, Activity Assay, Control, MANN-WHITNEY, Activation Assay, Stable Transfection, Expressing, Concentration Assay, Western Blot, Infection, Staining, Incubation

Journal: Microarrays

Article Title: Evaluation of Solid Supports for Slide- and Well-Based Recombinant Antibody Microarrays

doi: 10.3390/microarrays5020016

Figure Lengend Snippet: Evaluation of slide-based solid supports for antibody microarrays. The supports were compared with respect to blocking buffer, surface fouling, spot morphology and signal strength. ( A ) The slides (no antibodies printed) were first blocked and then incubated with crude, labelled serum and scanned after washing. Any observed signal intensity represents non-specific background binding. Representative scans of FAST, black MaxiSorp, Nexterion H, epoxy polymer and NHS glass slides are shown. The blocking agents tested are shown to the right; ( B ) Scanned microarray images of 14 × 8 antibody microarrays on epoxy glass, epoxy polymer, NHS glass, Nexterion H, black MaxiSorp, GAPSII, Nexterion P, Silane-Prep and NHS polymer slides. The printed antibody arrays were blocked and then incubated with crude, labelled serum before scanning.

Article Snippet: For manual processing, individual sub-arrays were created by using silicon incubation chambers (Schott) or a hydrophobic pen (Dako, Glostrup, Denmark) for slides that did not fit the incubation chambers (Silane-Prep (Sigma) and GAPSII (Corning)).

Techniques: Blocking Assay, Incubation, Binding Assay, Polymer, Microarray

Journal: Cell Communication and Signaling : CCS

Article Title: Dysregulated Gab1 signalling in triple negative breast cancer

doi: 10.1186/s12964-024-01542-9

Figure Lengend Snippet: Constitutive signalling in breast cancer cell lines. A MCF7, T-47D, SKBR3, MDA-MB-468, UACC-3199, MDA-MB-231 and Hs 578T cells were seeded and cultivated in phenol red-free RPMI 1640 for 24 h. On the following day, cells were lysed and proteins were separated by SDS-PAGE. After Western blotting, membranes were stained with specific antibodies against vimentin, E-cadherin, (p)Y EGFR, EGFR, (p)Y Gab1, Gab1, (p)T/Y ERK1/2, ERK1/2, (p)S Akt, Akt, (p)Y STAT3, STAT3 and tubulin. A representative result of n = 3 independent experiments is shown. B Expression or phosphorylation of (p)Y EGFR, EGFR, (p)Y Gab1, Gab1, (p)T/Y ERK1/2, (p)S Akt and (p)Y STAT3 from n = 3 independent experiments were quantified and normalised to tubulin expression. The highest phosphorylation or expression among the cell lines analysed was set to 100%. High expression or phosphorylation is visualised in dark red. For each cell type the normalised phosphorylation and expression strengths of all analysed entities are summed up and depicted in blue. C MDA-MB-468 or MDA-MB-231 cells were seeded on poly-L-lysine-coated glass cover slips and cultivated in phenol red-free RPMI 1640. After 24 h, cells were transfected with an expression vector for murine Gab1-GFP. On the following day, cells were placed into the incubation chamber of a laser scanning microscope. Imaging was performed after 30 min equilibration. Representative results of n = 3 independent experiments are shown

Article Snippet: Prior to usage, the temperature of the incubation chamber (PeCon, Erbach, Germany) and of the objective lens was adjusted to 37 °C and the atmosphere within the incubation chamber was set to 5% CO 2 . eGFP-Gab1 fusion proteins were excited using laser light of 488 nm.

Techniques: SDS Page, Western Blot, Staining, Expressing, Phospho-proteomics, Transfection, Plasmid Preparation, Incubation, Laser-Scanning Microscopy, Imaging

Journal: Cell Communication and Signaling : CCS

Article Title: Dysregulated Gab1 signalling in triple negative breast cancer

doi: 10.1186/s12964-024-01542-9

Figure Lengend Snippet: EGFR activity promotes constitutive Gab1 and MAPK phosphorylation in MDA-MB-468 cells. A MDA-MB-468 cells were seeded and cultivated in phenol red-free RPMI 1640 for 24 h. On the following day, cells were treated with DMSO or Gefitinib (3 µM) for 30 min. Subsequently, cells were lysed and proteins were separated by SDS-PAGE. After Western blotting, membranes were stained with specific antibodies against (p)Y Gab1, Gab1, (p)T/Y ERK1/2, ERK1/2, (p)S Akt, Akt, (p)Y STAT3, STAT3 and tubulin. A representative result of n = 3 independent experiments is shown. The results from ( A ) were quantified. The diagrams show the ratios of B (p)Y Gab1 to Gab1, C (p)T/Y ERK1/2 to ERK1/2, D (p)S Akt to Akt, and E (p)Y STAT3 to STAT3. Gab1, ERK1/2, Akt and STAT3 phosphorylation in DMSO-treated cells was normalised to 100% in each independent repetition of the experiment. Data are given as mean of three independent experiments ± SD. Student’s t-test: n.s. = non-significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001. F MDA-MB-468 cells were seeded on poly-L-lysine-coated glass cover slips and cultivated in phenol red-free RPMI 1640. After 24 h, cells were transfected with an expression vector for murine Gab1-GFP. On the following day, cells were placed into the incubation chamber of a laser scanning microscope and left for 30 min. Cells were treated with Gefitinib (3 µM) or DMSO for 30 min. Imaging was performed after treatment with Gefitinib or DMSO. Representative results of n = 3 independent experiments are shown

Article Snippet: Prior to usage, the temperature of the incubation chamber (PeCon, Erbach, Germany) and of the objective lens was adjusted to 37 °C and the atmosphere within the incubation chamber was set to 5% CO 2 . eGFP-Gab1 fusion proteins were excited using laser light of 488 nm.

Techniques: Activity Assay, Phospho-proteomics, SDS Page, Western Blot, Staining, Transfection, Expressing, Plasmid Preparation, Incubation, Laser-Scanning Microscopy, Imaging

Journal: Cell Communication and Signaling : CCS

Article Title: Dysregulated Gab1 signalling in triple negative breast cancer

doi: 10.1186/s12964-024-01542-9

Figure Lengend Snippet: MAPK and PI3K signalling are crucial for Gab1 plasma membrane recruitment in MDA-MB-468 cells. A MDA-MB-468 cells were seeded on poly-L-lysine-coated glass cover slips and cultivated in phenol red-free RPMI 1640. After 24 h, cells were transfected with an expression vector for either murine Gab1-GFP, murine Gab1-S552A-GFP or murine Gab1-ΔPH-GFP. On the following day, cells were placed into the incubation chamber of a laser scanning microscope. Imaging was performed after 30 min equilibration. Representative results of n = 3 independent experiments are shown. B MDA-MB-468 cells were seeded on poly-L-lysine-coated glass cover slips and cultivated in phenol red-free RPMI 1640. After 24 h, cells were transfected with an expression vector for murine Gab1-GFP. On the following day, cells were placed into the incubation chamber of a laser scanning microscope and left for 30 min. Cells were treated with DMSO, U0126 (10 µM) and/or LY294002 (40 µM) for 30 min. Imaging was performed before and after treatment. Representative results of n = 3 independent experiments are shown

Article Snippet: Prior to usage, the temperature of the incubation chamber (PeCon, Erbach, Germany) and of the objective lens was adjusted to 37 °C and the atmosphere within the incubation chamber was set to 5% CO 2 . eGFP-Gab1 fusion proteins were excited using laser light of 488 nm.

Techniques: Clinical Proteomics, Membrane, Transfection, Expressing, Plasmid Preparation, Incubation, Laser-Scanning Microscopy, Imaging

Journal: Cell Communication and Signaling : CCS

Article Title: Dysregulated Gab1 signalling in triple negative breast cancer

doi: 10.1186/s12964-024-01542-9

Figure Lengend Snippet: Gab1 is localised in the cytoplasm in PI3K and MAPK inhibition resistant MDA-MB-468 cells. MDA-MB-468 cells were seeded on poly-L-lysine-coated glass cover slips and cultivated in phenol red-free RPMI 1640. After 24 h, cells were transfected with an expression vector for murine Gab1-GFP. On the following day, cells were treated with DMSO, U0126 (10 µM) or LY294002 (40 µM). After 48 h cells were placed into the incubation chamber of a laser scanning microscope. Imaging was performed after 30 min equilibration. Representative results of n = 3 independent experiments are shown

Article Snippet: Prior to usage, the temperature of the incubation chamber (PeCon, Erbach, Germany) and of the objective lens was adjusted to 37 °C and the atmosphere within the incubation chamber was set to 5% CO 2 . eGFP-Gab1 fusion proteins were excited using laser light of 488 nm.

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Incubation, Laser-Scanning Microscopy, Imaging